T4 DNA LIGASE

CAS:      9015-85-4

Artikel Nr: P6402

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Properties

Synonyms:

RNA LIGASE, T4 PAHGE;

DNA LIGASE;

DNA LIGASE NAD-DEPENDENT;

DNA LIGASE T4;

DNA LIGASE, THERMOSTABLE;

EC 6.5.1.1;EC 6.5.1.2;

IUB: 6.5.1.1

CAS:      9015-85-4

EINECS:               232-770-0

Product Categories:       Enzyme

storage temp.:  −20°C

form:    buffered aqueous glycerol solution, 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50%. Glycerol, pH 7.4 @ 25°C

Reaction conditions: 1X T4 DNA Ligase Reaction Buffer

Specification: 16,000 U / 80,000 U

Concentration: 400,000 U/mL

Products components:  T4 DNA Ligase (400,000 U/mL) RM21501

10X T4 DNA ligase Reaction Buffer

Components of 10X T4 DNA ligase Reaction Buffer: 50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, pH7.5 @ 25°C

 

Thermal deactivation: 65°C heating 10 min

 

 

Safety

WGK Germany:               3

F:            10

TSCA:    Yes

 

Product Description

T4 DNA ligase catalyzes the formation of phosphodiester bonds between the 5′-phosphate and 3′-hydroxyl groups of double-stranded DNA or RNA. ATP is required as a cofactor for this reaction. This enzyme catalyzes ligation between sticky end molecules or between flat end molecules and also repairs single-stranded DNA cuts on double-stranded DNA or DNA-RNA hybrid duplexes. At the same time T4 DNA ligase closes these DNA substrate gaps. This product is suitable for ligation of restriction endonuclease sections, linkers, or adapters, etc. It can also be used for section repair and ligase-mediated RNA detection.

 

Product Sources

The T4 DNA Ligase gene of phage origin was recombinantly expressed in E. coli and purified through a multi-step purification process.

Uses

Forms an energy dependent phosphodiester linkage between the termini of adjacent polynucleotides of duplex DNA. For ligation of cloning vector and restriction insert fragments

General Description        T4 DNA Ligase catalyzes the formation of phosphodiester bonds between neighboring 3′-hydroxyl- and 5′-phosphate ends in double-stranded DNA. Single-stranded nicks in double-stranded DNA are also closed by T4 DNA Ligase.

 

Contents

T4 DNA Ligase, supplied with 10x concentrated ligation buffer that includes ATP.

 

Protocol

  • Add each reaction component (20 μL reaction system as an example) on ice as shown in the table below:
componentAmount to be added
10X T4 DNA Ligase Reaction Buffer2 μL
Vector DNA (4 kb)50 ng (0.02 pmol)
Insertion of DNA fragment (1 kb)37.5 ng (0.06 pmol)
T4 DNA Ligase1 μL
ddH2OUp to 20 μL

 

  • The reaction components were mixed and briefly centrifuged to collect the solution at the bottom of the tube.
  • For Sticky Ends substrates, react overnight at 16°C or ligate for 10 min at room temperature.
  • For Blunt Ends or single base protruding ends, ligate at 16°C overnight or at room temperature for 2 h (you can also choose a high concentration of T4 DNA Ligase for 10 min at room temperature).
  • (Optional) Heat at 65°C for 10 min to inactivate T4 DNA ligase.
  • Place the reaction product on ice and 1-5 μl of the ligand product was added to 50 μl of the sensory of the receptor cells. The transformation was performed.

Quality Control

The purity of the protein by SDS-PAGE was >95%.

No nucleic acid exonuclease, endonuclease or RNA enzyme activity.

PCR assays are free of host genome residues.

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